H1 histones bind to linker DNA and nucleosome core particles and facilitate the folding of chromatin into a more compact structure. Mammals contain seven nonallelic subtypes of H1, including testis-specific subtype H1t, which varies considerably in primary sequence from the other H1 subtypes. H1t is found only in pachytene spermatocytes and early, haploid spermatids, constituting as much as 55% of the linker histone associated with chromatin in these cell types. To investigate the role of H1t in spermatogenesis, we disrupted the H1t gene by homologous recombination in mouse embryonic stem cells. Mice homozygous for the mutation and completely lacking H1t protein in their germ cells were fertile and showed no detectable defect in spermatogenesis. Chromatin from H1t-deficient germ cells had a normal ratio of H1 to nucleosomes, indicating that other H1 subtypes are deposited in chromatin in place of H1t and presumably compensate for most or all H1t functions. The results indicate that despite the unique primary structure and regulated synthesis of H1t, it is not essential for proper development of mature, functional sperm.The histones are a family of basic proteins that are involved in organizing the DNA in the nuclei of eukaryotic cells into a compact structure called chromatin. There are five major classes of histones, the core histones H2A, H2B, H3, and H4 and the linker histone H1. Two molecules of each of the core histones constitute the protein octamer of the nucleosome core particle. H1 histones bind to DNA in the nucleosome core particle and to the linker DNA between nucleosomes. These interactions are thought to facilitate the folding of nucleosomes into the 30-nm chromatin fiber and higher-order chromatin structures (39,42). Interactions between histones and DNA would be expected to modulate gene activity, and recent evidence clearly shows that both the core histones and H1 can have a profound effect on transcription (reviewed in references 18, 43, and 44).Among the five classes of histones, the H1 histones exhibit the most diversity. For mice seven H1 subtypes have been described (24, 25), including the "somatic" subtypes H1a through H1e, the replacement subtype H1 o , and the testisspecific linker histone H1t. These seven H1 subtypes are also present in humans, and the genomic organization of the genes encoding the H1 subtypes in humans appears to be very similar to that in mice (12,40).Within the H1 family of proteins, the testis-specific H1t subtype is unique in that it is the only member exhibiting a truly tissue-specific pattern of expression. Although the other six H1 subtypes display distinct patterns of expression during differentiation and development, they are all expressed in numerous tissues (24-26). On the other hand, the H1t gene is transcribed exclusively in mid-and late-pachytene spermatocytes (10, 13, 16), and H1t protein is found only in pachytene spermatocytes and early, haploid spermatids (11,17,29), in which it constitutes up to 55% of the total H1 linker histone in chromatin (29). M...

DNA polymerase (pol ) is a conserved Y family enzyme that is implicated in translesion DNA synthesis (TLS) but whose cellular functions remain uncertain. To test the hypothesis that pol performs TLS in cells, we compared UV-induced mutagenesis in primary fibroblasts derived from wild-type mice to mice lacking functional pol , pol , or both. A deficiency in mouse DNA polymerase (pol ) enhanced UV-induced Hprt mutant frequencies. This enhanced UV-induced mutagenesis and UV-induced mutagenesis in wild-type cells were strongly diminished in cells deficient in pol , indicating that pol participates in the bypass of UV photoproducts in cells. Moreover, a clear strand bias among UV-induced base substitutions was observed in wild-type cells that was diminished in pol -and pol -deficient mouse cells and abolished in cells deficient in both enzymes. These data suggest that these enzymes bypass UV photoproducts in an asymmetric manner. To determine whether pol status affects cancer susceptibility, we compared the UV-induced skin cancer susceptibility of wild-type mice to mice lacking functional pol , pol , or both. Although pol deficiency alone had no effect, UV-induced skin tumors in pol -deficient mice developed 4 weeks earlier in mice concomitantly deficient in pol . Collectively, these data reveal functions for pol in bypassing UV photoproducts and in delaying the onset of UV-induced skin cancer. The most compelling evidence for a role in translesion synthesis (TLS) is with pol . pol can efficiently bypass a cis-syn thymine-thymine dimer (6, 7), it localizes at replication foci after UV irradiation of mammalian cells (8), and its inactivation in XPV patients (9, 10) increases UV light-induced mutagenesis and skin cancer (reviewed in refs. 4 and 11). Increased UVinduced mutagenesis associated with pol deficiency indicates that mutagenic bypass of UV photoproducts can be catalyzed by other polymerases. One candidate is pol (2), which can bypass a thymine-thymine dimer in vitro (12) and is required for a large proportion of UV mutagenesis in vivo (2,13,14). Another candidate is pol , which physically interacts with pol and colocalizes with pol at replication foci after UV irradiation (8).Two hypotheses have been put forth regarding the outcome of putative UV photoproduct bypass by pol . One suggests that bypass should be mutagenic because of pol -catalyzed misinsertion of nucleotides opposite photoproducts (15, 16). pol is renowned for high rates of misinsertion of nucleotides opposite template pyrimidines during DNA synthesis in vitro (reviewed in ref.3). However, a study of UV-induced mutagenesis in cultured 293T cells in which pol expression was decreased by using siRNA concluded that pol has no significant role in UV lesion bypass and mutagenesis (17). A second hypothesis is that pol bypass could be antimutagenic for UV-induced C to T transition mutations. This antimutagenic hypothesis derives from a pol preference for inserting dGMP opposite template T, leading to the suggestion (18, 19) that synthesis by pol could b...

Posttranslational modifications and remodeling of nucleosomes are critical factors in the regulation of transcription. Higher-order folding of chromatin also is likely to contribute to the control of gene expression, but the absence of a detailed description of the structure of the chromatin fiber has impaired progress in this area. Mammalian somatic cells contain a set of H1 linker-histone subtypes, H1 (0) and H1a to H1e, that bind to nucleosome core particles and to the linker DNA between nucleosomes. To determine whether the H1 histone subtypes play differential roles in the regulation of gene expression, we combined mice lacking specific H1 histone subtypes with mice carrying transgenes subject to position effects. Because position effects result from the unique chromatin structure created by the juxtaposition of regulatory elements in the transgene and at the site of integration, transgenes can serve as exquisitely sensitive indicators of chromatin structure. We report that some, but not all, linker histones can attenuate or accentuate position effects. The results suggest that the linkerhistone subtypes play differential roles in the control of gene expression and that the sequential arrangement of the linker histones on the chromatin fiber might regulate higher-order chromatin structure and fine-tune expression levels.

Tumor-initiating cell (TIC) is a subpopulation of cells in tumors that are responsible for tumor initiation and progression. Recent studies indicate that hepatocellular carcinoma-initiating cells (HCICs) confer the high malignancy, recurrence and multi-drug resistance in hepatocellular carcinoma (HCC). In this study, we found that Icaritin, a prenylflavonoid derivative from Epimedium Genus, inhibited malignant growth of HCICs. Icaritin decreased the proportion of EpCAM-positive (a HCICs marker) cells, suppressed tumorsphere formation in vitro and tumor formation in vivo. We also found that Icaritin reduced expression of Interleukin-6 Receptors (IL-6Rs), attenuated both constitutive and IL-6-induced phosphorylation of Janus-activated kinases 2 (Jak2) and Signal transducer and activator of transcription 3 (Stat3), and inhibited Stat3 downstream genes, such as Bmi-1 and Oct4. The inhibitory activity of Icaritin in HCICs was augmented by siRNA-mediated silencing of Stat3 but attenuated by constitutive activation of Stat3. Taken together, our results indicate that Icaritin is able to inhibit malignant growth of HCICs and suggest that Icaritin may be developed into a novel therapeutic agent for effective treatment of HCC.